When cells respond to extracellular stimuli, various proteins in the cells are involved in the response. Thus, agonists and/or antagonists targeting such proteins may be used as lead compounds for novel drugs.
Conventional methods of searching for agonists and/or antagonists to a protein include searching by use of physical binding to the target protein as indicator and searching by use of capacity to induce a biological reaction involving the target protein (functional induction capacities) as indicator.
In the method of searching by use of physical binding to the target protein as indicator, compounds from a compound library (e.g., a combination library) are examined for strength of binding to the target protein, number of bound molecules per molecule thereof and so forth, and then high-affinity compounds to the target protein above a certain reference affinity are screened.
On the other hand, in the method using functional induction capacity for the target protein, namely, a biological reaction involving the target protein, as indicator, compounds from a compound library (e.g., a combination library) are contacted with cells expressing the target protein and the resulting variations in the biological reaction (for example, qualitative variations such as initiation and termination of the biological reaction, or quantitative variations such as increased or decreased degree of the biological reaction) are examined as indicator. To carry out the method using functional induction capacity as indicator, it is required to establish an assay system specific to an individual target protein. As a consequence, when the effect of the compound is analyzed, for example, for living cells, there are such limitations that its reliability and experimental accuracy may depend on the assay system.
Agonists and/or antagonists to the protein are identified and the identified compounds can be used as lead compound for a novel drug. In screening candidate compounds for a novel drug, it is useful to see how the conformation of the target protein is varied by their binding to the target protein. It is because such information of conformational variations may provide a clue to the design of a candidate drug having a more appropriate structure.
However, in either case of physical binding or functional induction capacity used as indicator, the state of binding of the compound to the protein is not directly observed. Therefore, the structure and function of the compound is speculated by analogy from the binding capacity or functional induction capacity for the target protein provided as indirect observation. Simple determination of the presence or absence of physical binding or functional induction capacity may hardly reveal the real state of interaction between the target protein and the compound and may not always provide a clear guide for drug design.
In order to eliminate such problems presented by conventional assay methods, an assay method utilizing nuclear magnetic resonance (NMR) has been published in which binding of a compound to the functional site of a target protein is confirmed by NMR (Shuker S. B. et al., Science (1996) 274, 1531-1534) (JP-A-2001-321192). In this technique of prior art, such a case that the compound is non-specifically bound to a non-functional site of the target protein can be excluded, and lead compounds can be thus identified more efficiently than in the conventional assay of physical binding which has a difficulty in distinguishing between nonspecific binding and specific binding. However, the technique is not suitable to measure a structural variation itself in the protein, though it enables the binding site to be identified. Therefore, this technique is effective to design inhibitors capable of binding to the active site of the target protein, but is not so effective to design agonistic agents (e.g., agonists) capable of inducing the activity of the protein. Since identification of an agonistic lead compound requires to screen compounds of interest by seeing if they bind properly to the binding site on the target protein to its substrate and further induce the active structure of the target protein, this conventional technique has not been adequate to identify the agonistic lead compound.
It can be determined by structural analysis based on X-ray crystallography if a compound can induce the active structure of a target protein. However, the X-ray crystallographical structural analysis is not suitable for efficient screening since it needs enormous amounts of labor and time to crystallize many compounds together with the target protein and analyze the conformation of as many crystals. Consequently, a simple and rapid method to determine a structural change in the target protein, which may occur when an arbitrary compound is contacted with the target protein and bound thereto, and a computer program to carry out the method are needed.
Patent Document 1: JP-A-2001-321192.
Non-Patent Document 1: Shuker S. B. et al., Science (1996) 274, 1531-1534.